Plant expression systems have been developed as a production platform for therapeutic proteins in the past two decades. Plants have some advantages over other expression systems, such as mammalian cell culture and bacterial fermentation. The application of plant systems means a lower cost of production and large-volume production, and cultivation is much less expensive and easier without sterile conditions of cell culture. Like mammalian systems, plant expression systems have the advantage of being able to produce active forms of complex proteins with post-translational modifications, such as glycosylation, which are necessary for human therapeutic proteins for correct function in vivo. Plant systems are also free of human pathogens potentially associated with mammalian cell cultures.
Although much work has been done with transgenic plants, their creation is time-consuming and labor-intensive. Plant viral vectors have emerged as the most efficient approach to achieving more rapid and higher-level expression of recombinant proteins, although protein expression is transient. Viral vectors systems take advantage of high levels of replication and maximum levels of foreign gene expression in a short time period from an engineered viral genome, with results within a week or two post-inoculation. A number of different plant viruses have been developed into protein production vectors, the most commercially useful being tobacco mosaic virus (TMV) of the Tobamovirus family and potato virus X (PVX) of the Potexvirus family.
Over the last two decades, plant virus-based expression systems have been successfully developed and utilized for high-yield production of heterologous proteins in plants. Viral vectors as transient gene expression systems provide increased speed and flexibility during early phases of experimentation. However, the potential widespread use of recombinant viruses raises concerns about possible risks to the environment. The bio-safety issues have to be considered to control the spread of the genetically engineered virus from experimental plants to susceptible wild plants. Intact viral vectors have the potential to spread and infect non-target plants, but replication-defective or movement-defective viruses avoid these problems. These deleted viral vectors can be safely used in the laboratory and, in large scale application, can be used to inoculate an entire greenhouse at once. In the field, it may be possible to achieve high expression in transgenic plants carrying an inducible virus as a transgene. In all of these cases, deleted virus vectors would be greatly preferred over full virus vectors for their lower environmental risk. However, one disadvantage of the deleted virus approach is that the vector cannot spread past the originally inoculated cells.
Several innovations have led to dramatic improvements in plant viral vectors. The early versions of these vectors cited used in vitro transcription to create infectious RNA, which is expensive and not amenable to large scale production in contrast to a more recent method named “agroinfection” (Gleba et al., 2005). Agroinfection involves syringe or vacuum infiltration of an Agrobacterium tumefaciens suspension harboring T-DNA carrying the viral genome into plant leaves, resulting in the local transformation of the infiltrated leaf with the cDNA form of the virus as a part of the T-DNA of the Ti plasmid. Agrobacterium infects each cell in the inoculated zone and inserts its T-DNA into the plant chromosome of each cell. A plant promoter placed upstream of the viral cDNA induces the transcription of viral genome in the plant nucleus and viral RNA is transported to cytoplasm for viral replication. Agroinfection results in almost 100% of the plant cells being infected transformed by Agrobacterium in the infiltrated zone. Therefore, agroinoculation also gives a preview as to how expression would look in a permanently transgenic plant. For both agroinoculation and transgenic use, systemic spread becomes an unnecessary property.
Agroinfection was developed originally for DNA plant viruses. As DNA viruses have disadvantages for foreign sequences insertion, RNA viruses were introduced into agroinfection system and developed with a number of different RNA viruses. Another development was the use of RNA silencing suppressors to increase expression. For example, a recently developed TMV vector, driven by a 35S promoter in a binary vector, was delivered via agroinfection along with an Agrobacterium culture carrying a 35S-driven p19 suppressor. This system produced 0.6-1.2 mg of recombinant protein per gram of infiltrated plant tissue, which is 10-25 times higher than the 35S promoter driven transient expression systems (Lindbo, 2007a).
Agroinfection allows the replacement of the MP and/or CP genes of vector viruses with heterologous sequences in some virus species. Tobacco mosaic virus (TMV) lacking the CP gene has been used to produce large amounts of foreign proteins and agroinfection greatly increased infectivity of the TMV cDNA, since every cell in the infiltrated area contained the TMV transgene in its nucleus. In the potato virus X (PVX) replacement virus vector, both the triple gene block (TGB) and coat protein (CP) viral genes were removed, leaving only the replicase gene, and were replaced with GFP. The expression levels of GFP from this vector were about 2.5-fold higher than that of full-length PVX vector with the GFP encoding sequence between the triple gene block and the CP genes. Removal of the movement proteins prevents systemic movement of TMV and PVX in above examples and inhibits the spread of the genetically modified virus, which is positive from the biosafety point of view.
Agrobacterium infiltration-mediated transient expression can be greatly enhanced by suppression of gene silencing. An RNA silencing suppressor (such as P19 encoded by tomato bushy stunt virus or HcPro expressed by potato virus A) is co-inoculated in a separate strain of Agrobacterium along with the Agrobacterium carrying the viral cDNA. Using this approach, highly efficient production of GFP from a TMV-based vector was achieved with up to 100-fold increase of the overexpression level (Lindbo, 2007a). As well, potexvirus expression was greatly increased (Komorova et al., 2006). Both of these viral vectors expressed GFP efficiently in the absence of GFP; however, the addition of GFP greatly increased this efficiency.
Foxtail Mosaic Virus (FoMV)
Foxtail mosaic virus (FoMV) is a member of the genus Potexvirus. Potexvirus is a large group of flexous and filamentous plant viruses with a single-stranded, positive-sense genomic RNA, with a cap structure at the 5′ terminus and a poly-(A) tail at the 3′ terminus. The FoMV genome structure resembles that of PVX, the type species of the genus Potexvirus, and the gene functions are presumed to be similar as well. The genome of FoMV contains five open reading frames (ORFs), and two subgenomic promoters directing transcription of subgenomic RNAs 1 and 2 (sgRNA1 and sgRNA2). The genomic RNA allows the expression of ORF1 encoding for the RNA-dependent RNA polymerase (RdRP) with methyltransferase, helicase, and polymerase motifs. ORF2, 3 and 4 code for the triple gene block (TGB) proteins TGB1, TGB2 and TGB3, which are required for virus cell-to-cell movement. ORF2 codes for a multifunctional protein that has RNA helicase activity, promotes translation of viral RNAs, increases plasmodesmal size exclusion limits, and acts as a suppressor of RNA-mediated post-transcriptional gene silencing (PTGS). ORF5 encodes the coat protein, which is required for viral encapsidation and long distance movement. FoMV has a broad host range, infecting 56 species of the Gramineae and at least 35 dicot species. The sequence of FoMV genomic RNA was first published in 1991. Infectious full-length clones were constructed based on the same FoMV isolate and some corrections to the published sequence were noted. The significant difference between the gene organizations of FoMV and PVX is the presence of ORF 5A upstream of the CP gene in FoMV. ORF 5A initiates 143 nts upstream of the CP and extends the reading frame of CP gene. The 5A protein was produced in vivo, but it was not required for either replication or productive infection of plants. Recently, the revised full-length sequence of Foxtail mosaic virus clone was published in 2008 (Bruun-Rasmussen et al.), and reveals a triple gene block structure similar to potato virus.
Foundational potexvirus vector work was done first not with FoMV but with PVX, the type species of the genus Potexvirus. PVX was engineered to express reporter proteins such as GFP and GUS, which were cloned just upstream of the CP gene and expressed from a duplicated copy of the coat protein (CP) subgenomic promoter. The reporter protein is translated from a sgRNA separate from the other viral proteins. Because PVX has a linear helical capsid, rather than an icoshedral capsid, the longer than wild type recombinant viral genome can still be encapsidated into infectious virus particles. However, GUS encoding sequence was deleted, because of recombination between the homologous sequences of the duplicated subgenomic promoters (81 nt).
The potexvirus replicase is the only protein translated directly from the full-length genomic RNA, but other viral proteins are translated from 3′ coterminal subgenomic RNAs (sgRNAs). The two sgRNAs of approximately 2.1 and 0.9 kb in length have their 5′ termini upstream of the TGB and CP genes, respectively. The integrity of subgenomic promoter is very important for the accumulation of subgenomic RNA and target protein. However, the boundaries of sgRNA promoters have not been delineated for FoMV.
Therefore, what is needed in either a greenhouse or field setting is a viral vector which expresses protein at a very high level and yet is intrinsically crippled, greatly facilitating decontamination and lowering environmental risk. As well, an inducible version of such a viral vector which expresses an exogenous gene at high levels in the presence of a silencing suppressor, and expresses the exogenous gene at negligible levels in the absence of a silencing suppressor would further lower environmental risk and would facilitate the manipulation of plant material transformed with such a viral vector, such as in the production of transgenic plants.